HIV Drug Resistance in Adults Initiating or Reinitiating Antiretroviral Therapy in Uruguay—Results of a Nationally Representative Survey, 2018–2019

The first nationally representative cross-sectional HIV drug resistance (HIVDR) survey was conducted in Uruguay in 2018–2019 among adults diagnosed with HIV and initiating or reinitiating antiretroviral therapy (ART). Protease, reverse transcriptase, and integrase genes of HIV-1 were sequenced. A total of 206 participants were enrolled in the survey; 63.2% were men, 85.7% were >25 years of age, and 35.6% reported previous exposure to antiretroviral (ARV) drugs. The prevalence of HIVDR to efavirenz or nevirapine was significantly higher (OR: 1.82, p < 0.001) in adults with previous ARV drug exposure (20.3%, 95% CI: 18.7–22.0%) compared to adults without previous ARV drug exposure (12.3%, 11.0–13.8%). HIVDR to any nucleoside reverse transcriptase inhibitors was 10.3% (9.4–11.2%). HIVDR to ritonavir-boosted protease inhibitors was 1.5% (1.1–2.1%); resistance to ritonavir-boosted darunavir was 0.9% (0.4–2.1%) among adults without previous ARV drug exposure and it was not observed among adults with previous ARV drug exposure. Resistance to integrase inhibitors was 12.7% (11.7–13.8%), yet HIVDR to dolutegravir, bictegravir, and cabotegravir was not observed. The high level (>10%) of HIVDR to efavirenz highlights the need to accelerate the transition to the WHO-recommended dolutegravir-based ART. Access to dolutegravir-based ART should be prioritised for people reporting previous ARV drug exposure.


Introduction
HIV drug resistance negatively impacts the effectiveness of antiretroviral (ARV) drugs in preventing and treating HIV infection. HIV drug resistance increases the number of HIV/AIDS-associated deaths, new HIV infections, and antiretroviral therapy (ART) programme costs [1]. Therefore, the surveillance, prevention and control of HIV drug resistance are essential to achieving the 95-95-95 targets and the elimination of AIDS as a public health threat by 2030 [2]. The World Health Organization (WHO) recommends the surveillance of HIV drug resistance among adults initiating and reinitiating ART to inform the selection of effective first-line ART regimens and adequate prophylaxis regimens [3].
The prevalence of HIV drug resistance among adults initiating and reinitiating ART has increased in Latin America and the Caribbean: An annual increase of 11% (95% CI: 5-18%) in the odds of HIV drug resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI) was reported from 2007 to 2016 [4]. Furthermore, significantly higher levels of NNRTI resistance (OR: 3.4, 95% CI: 1.8-6.9%, p < 0.0001) have been reported among adults with previous ARV drug exposure compared with those without previous ARV drug exposure [4]. Several low-and middle-income countries have reported a nationally representative prevalence of HIV drug resistance to efavirenz (EFV) or nevirapine (NVP) above 10% among adults initiating and reinitiating ART [5], the WHO-recommended threshold to urge transition to a non-NNRTI-containing regimen [6].
This report presents the findings of the first nationally representative survey to estimate the prevalence of HIV drug resistance among adults initiating and reinitiating ART in Uruguay from October 2018 to October 2019. At the time of the survey, 9240 adults living with HIV were receiving ART, and <1000 adults were newly infected with HIV annually [7].

Study Design
The Ministry of Health of Uruguay implemented a cross-sectional nationally representative survey following the WHO-recommended methods [3]. Twenty-two public and private clinics provided ART in Uruguay at the time of the survey. Eleven ART clinics were selected as survey sites. These clinics served 94% of all adults initiating and reinitiating ART in Uruguay. The excluded eleven ART clinics had less than ten adults initiating and reinitiating ART per year. The sample size was calculated using the WHO sample size calculator tool and utilised a finite population correction [8]. The following assumptions were used to calculate the sample size: (a) a prevalence of HIV drug resistance to EVF or NVP of 10% among adults initiating and reinitiating ART, (b) an absolute precision of 5%, (c) a genotyping failure rate of 20%, (d) 840 adults initiating and reinitiating ART in 2016, (e) a proportion of previous ARV drug exposure of 25%, and (f) a proportion of adults initiating and reinitiating ART with regimens that included NNRTIs of 80%. Using these assumptions, the total sample size was 239, and it was distributed among survey sites proportionally to the number of adults initiating or reinitiating ART at each of the eleven clinics selected to participate in the survey.

Participant Enrolment
Adults ≥ 18 years of age diagnosed with HIV initiating or reinitiating ART were eligible for survey enrolment. Eligible individuals who provided verbal informed consent were consecutively enrolled. Deidentified demographic and clinical data were abstracted from clinical records. Previous ARV drug exposure was classified as pre-exposure prophylaxis, post-exposure prophylaxis, prevention of mother-to-child transmission, discontinuation of prior ART, or a combination of these exposures.

Laboratory Procedures
Whole blood specimens were collected by venipuncture in EDTA-containing tubes from participants before initiating or reinitiating ART. Plasma was separated within 12 h of collection; all plasma specimens were stored at −80 • C at the National HIV-AIDS Reference Laboratory of Uruguay until the completion of the specimen collection period (October 2019). Plasma specimens were shipped on dry ice to the Centre for Research in Infectious Diseases of the National Institute of Respiratory Diseases in Mexico City, a WHO-designated regional laboratory for HIV drug resistance testing.

Data Analysis
Five pairs of HIV sequences with a small genetic distance (<0.5%) and without epidemiological link were detected. Therefore, following the WHO/HIV ResNet Laboratory Operational Framework [14], one sequence was excluded from each pair. Thus, 204 reverse transcriptase and protease sequences and 205 integrase sequences were used for the analysis. HIV drug resistance was predicted using the Stanford HIV drug resistance database (HIVdb v 8.9-1) tool [15,16]. Sequences with a penalty score ≥ 15 were considered resistant to a given ARV drug. HIV subtype was assigned using the REGA HIV-1 subtyping tool (v3.46) [17].
Weighted statistical analysis was performed according to WHO recommendations [3], using STATA 15.1 (StataCorp, College Station, TX, USA). The weights were calculated based on the estimated eligible population and the number of individuals enrolled in each survey site. The analysis of HIV drug resistance was weighted considering the genotyping success rate. Odds ratios were calculated by logistic regression accounting for survey design and weights. Statistical significance was assessed at the 0.05 level.
An HIV transmission network analysis was performed to identify and characterise transmission clusters. Sequences were aligned by codons using Mega v11 [18]. Phylogenetic trees were constructed using the maximum likelihood method based on the General Time Reversible model with 1000 bootstrap repetitions, including reference sequences for HIV subtypes obtained from the Los Alamos HIV Sequences Database (www.hiv.lanl.gov, accessed on 2 August 2022). The trees were visualised and coloured in Mega v11 [18]. Putative transmission links were defined and resolved between individuals whose HIV sequences had a genetic distance <1.5% using HIV-TRACE [19].
Resistance to the first-generation INSTIs was 14.9% (95% CI: 14.1-15.6%) in adults without previous exposure to ARV drugs. In adults with previous exposure to ARV drugs, resistance to first-generation INSTIs was 8.8% (95% CI: 6.6-11.5%). None of the people with previous ARV drug exposure and resistance to first-generation INSTIs reported receiving INSTI-based ART. The prevalence of the integrase mutation G163RK was 15.2% among adults without previous exposure to ARV drugs and 8.2% among those with previous exposure to ARV drugs (Table 4). This mutation was detected in all cases with resistance to first-generation INSTI. The cases in which G163KR was observed had infection with HIV-1 subtype F (34.6%), F1/B recombinants (34.6%), or other F recombinants (30.7%) ( Table 2).  NNRTI-based ART regimens were the most frequently prescribed (55.1%, 95% CI: 54-56.1%), followed by dolutegravir (DTG)-based regimens (29.4%, 95% CI: 28.4-30.4%), and PI/r-based regimens (11.7%, 95% CI: 11.0-12.6%) ( Table 1). Among people initiating and reinitiating NNRTI-based ART, 15.9% (95% CI: 14.4-17.5%) had resistance to EFV or NVP (Table 5). The prevalence of resistance to ATV/r or LPV/r was 4.8% among individuals initiating and reinitiating ATV/r-or LPV/r-based ART. Among those initiating and reinitiating DTG-based ART, none had DTG resistance.   Concerning HIV transmission, six clusters of two to three individuals were observed ( Figure 1). Five of the clusters corresponded to people without previous ARV drug exposure. Three groups had exclusively male individuals. Four clusters were from individuals residing in Montevideo, and one included individuals from Montevideo and Maldonado.

Discussion
In Uruguay, the prevalence of HIV drug resistance to EFV and NVP was high among people initiating and reinitiating ART. As reported in other Latin American countries [10,[22][23][24][25][26], the observed prevalence was greater than 10%, underscoring the need to accelerate the transition to the WHO-recommended DTG-based first-line ART [27,28]. Uruguay included DTG in the national ART guidelines in 2018. Therefore, at the time of the survey, DTG-based regimens were available in the country, but only 29.4% of those initiating and reinitiating ART received these regimens.
The prevalence of HIV drug resistance to EFV or NVP was significantly higher among individuals with previous ARV drug exposure than those without ARV drug exposure. Therefore, access to DTG-based regimens should be prioritised for people reporting previous exposure to ARV drugs. In addition, retention in care must be improved [27,29], and adequate support for treatment adherence must be provided to prevent HIV drug resistance among people on ART, thus minimising the risk of transmission of HIV-resistant variants [27,29].
The resistance to the first-generation INSTI in Uruguay was attributed to the G163KR mutation, which is polymorphic in HIV-1 subtype F [15,16] and has been reported in high prevalence among BF recombinant virus circulating among INSTI-naive individuals [30]. Indeed, in Uruguay, the cases with G163KR mutation were INSTI-naive and had infection with subtype F, BF recombinants, or other F recombinants. The G163KR mutation has not been associated with HIV resistance to second-generation INSTIs. Therefore, an impact on WHO-recommended DTG-based ART is not expected.
Several clusters of HIV transmission were observed in this survey, including three clusters with male individuals only. WHO recommends oral pre-exposure prophylaxis (PrEP) as an additional prevention choice for HIV-negative adults at a high risk of HIV acquisition [27]. Recommended PrEP regimens contain TDF with or without FTC or 3TC [27]. In Uruguay, among people without previous exposure to ARV drugs, the prevalence of HIV drug resistance to TDF was low (3.1%), and resistance to FTC or 3TC was not observed. These results support using these ARV drugs for PrEP regimens in the country.
Finally, approximately one-third of adults initiating and reinitiating ART with CD4 lymphocyte count had less than 200 cells/µL. Therefore, it is relevant to strengthen the interventions for early HIV diagnosis, including self-testing, index testing, and assisted partner notification [31,32], timely linkage to treatment, and retention in care [27]. In addition, comprehensive management of advanced HIV disease should be provided at the healthcare facilities in the country [33].
A strength of the survey is that it was carried out following WHO recommendations, and the results can be considered nationally representative estimates of HIV drug resistance among adults initiating and reinitiating ART in Uruguay. A limitation was that previous exposure to ARV drugs was self-reported and may have been underreported. Another limitation was that the enrolment was extended beyond the planned 6-month period to achieve the sample size.
In conclusion, this survey generated relevant, actionable data to inform public health interventions in Uruguay. First, accelerating the transition to DTG-based first-line ART as the preferred option at the national level, and prioritising access to this regimen for people with previous exposure to ARV drugs, will be key to minimising the impact of HIV drug resistance on HIV epidemic control goals. Second, HIV drug resistance should not be considered a barrier to scale-up oral pre-exposure prophylaxis for HIV. Third, retention in care and adequate support for ART adherence must be maximised to prevent HIV drug resistance. Finally, interventions are needed to ensure early HIV diagnosis, timely linkage to treatment, and retention in care.